RESUMEN
Escherichia coli has long been the primary prokaryotic host for the synthesis of heterologous proteins. Recent advances have been made in the expression of complex proteins as soluble, functional molecules, complete with prosthetic groups, disulfide bonds, and quaternary structure. The development of alternative promoter and induction strategies has improved the options available for manipulating the expression conditions, which are frequently critical to soluble yield.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Biotecnología , Disulfuros/metabolismo , Expresión Génica , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/genética , Regiones Promotoras GenéticasRESUMEN
Hydroxylamine-induced mutations in the virA gene of Agrobacterium tumefaciens that do not require the plant phenolic-inducing compound acetosyringone for vir regulon induction were isolated. The isolation was based on the activation of both virB::lacZ and virE::cat fusions by mutant virA loci in the absence of acetosyringone. Three of these virA(Ais) (acetosyringone-independent signaling) mutants were characterized. All three mutants expressed a virB::lacZ fusion at high levels in the absence of acetosyringone. One virA (Ais) mutant, virA112, exhibited vir gene expression in the absence of inducing monosaccharides and acidic growth conditions, both of which are normally required for vir gene induction. The phenotype of the virA112 mutant resulted from a glycine to glutamic acid change near His-474, the site of VirA autophosphorylation.
Asunto(s)
Agrobacterium tumefaciens/genética , Proteínas Bacterianas/genética , Mutación , Factores de Virulencia , Acetofenonas/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Análisis Mutacional de ADN , ADN Bacteriano , Genes Bacterianos , Glicina/química , Datos de Secuencia Molecular , Fenotipo , Fosforilación , PlásmidosRESUMEN
The genetic organization of the Pseudomonas aeruginosa acetyl coenzyme A carboxylase (ACC) was investigated by cloning and characterizing a P. aeruginosa DNA fragment that complements an Escherichia coli strain with a conditional lethal mutation affecting the biotin carboxyl carrier protein (BCCP) subunit of ACC. DNA sequencing and RNA blot hybridization studies indicated that the P. aeruginosa accB (fabE) homolog, which encodes BCCP, is part of a 2-gene operon that includes accC (fabG), the structural gene for the biotin carboxylase subunit of ACC. P. aeruginosa homologs of the E. coli accA and accD, encoding the alpha and beta subunits of the ACC carboxyltransferase, were identified by hybridization of P. aeruginosa genomic DNA with the E. coli accA and accD. Data are presented which suggest that P. aeruginosa accA and accD homologs are not located either immediately upstream or downstream of the P. aeruginosa accBC operon. In contrast to E. coli, where BCCP is the only biotinylated protein, P. aeruginosa was found to contain at least three biotinylated proteins.
Asunto(s)
Acetil-CoA Carboxilasa/genética , Ligasas de Carbono-Nitrógeno , Proteínas Portadoras/genética , Genes Bacterianos , Ligasas/genética , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Acetil-CoA Carboxilasa/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/biosíntesis , Clonación Molecular , Cartilla de ADN , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , Escherichia coli/enzimología , Escherichia coli/genética , Ligasas/biosíntesis , Datos de Secuencia Molecular , Operón , Plásmidos , Reacción en Cadena de la Polimerasa , ARN Bacteriano/biosíntesis , ARN Bacteriano/aislamiento & purificación , ARN Mensajero/biosíntesis , ARN Mensajero/aislamiento & purificación , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
The nac (nitrogen assimilation control) gene from Klebsiella aerogenes, cloned in a low-copy-number cloning vector, restored the ability of K. aerogenes nac mutants to activate histidase and repress glutamate dehydrogenase formation in response to nitrogen limitation and to limit the maximum expression of the nac promoter. When present in Salmonella typhimurium, the K. aerogenes nac gene allowed the hut genes to be activated during nitrogen-limited growth. Thus, the nac gene encodes a cytoplasmic factor required for activation of hut expression in S. typhimurium during nitrogen-limited growth.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Histidina/metabolismo , Klebsiella pneumoniae/genética , Nitrógeno/fisiología , Clonación Molecular , ADN Bacteriano/genética , Prueba de Complementación Genética , Operón , Mapeo Restrictivo , Salmonella typhimurium/genéticaRESUMEN
A positive, genetic selection against the activity of the nitrogen regulatory (NTR) system was used to isolate insertion mutations affecting nitrogen regulation in Klebsiella aerogenes. Two classes of mutation were obtained: those affecting the NTR system itself and leading to the loss of almost all nitrogen regulation, and those affecting the nac locus and leading to a loss of nitrogen regulation of a family of nitrogen-regulated enzymes. The set of these nac-dependent enzymes included histidase, glutamate dehydrogenase, glutamate synthase, proline oxidase, and urease. The enzymes shown to be nac independent included glutamine synthetase, asparaginase, tryptophan permease, nitrate reductase, the product of the nifLA operon, and perhaps nitrite reductase. The expression of the nac gene was itself highly nitrogen regulated, and this regulation was mediated by the NTR system. The loss of nitrogen regulation was found in each of the four insertion mutants studied, showing that loss of nitrogen regulation resulted from the absence of nac function rather than from an altered form of the nac gene product. Thus we propose two classes of nitrogen-regulated operons: in class I, the NTR system directly activates expression of the operon; in class II, the NTR system activates nac expression and the product(s) of the nac locus activates expression of the operon.
Asunto(s)
Sistemas de Transporte de Aminoácidos , Proteínas de Escherichia coli , Regulación Bacteriana de la Expresión Génica , Klebsiella pneumoniae/genética , Nitrógeno/fisiología , Proteínas de Saccharomyces cerevisiae , Asparaginasa/metabolismo , Glutamato Deshidrogenasa/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Histidina Amoníaco-Liasa/metabolismo , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mutación , Nitrato-Reductasa , Nitrato Reductasas/metabolismo , Operón , Factor sigma/fisiología , Ureasa/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
We initiated these studies to help clarify the roles of heme, delta-aminolevulinic acid (ALA), hemA, and hemM in Escherichia coli heme synthesis. Using recombinant human hemoglobin (rHb1.1) as a tool for increasing E. coli's heme requirements, we demonstrated that heme is a feedback inhibitor of heme synthesis. Cooverexpression of rHb1.1 and the hemA-encoded glutamyl-tRNA (GTR) reductase increased intracellular levels of ALA and heme and increased the rate of rHb1.1 formation. These results support the conclusion that heme synthesis is limited by ALA (S. Hino and A. Ishida, Enzyme 16:42-49, 1973; W. K. Philipp-Dormston and M. Doss, Enzyme 16:57-64, 1973) and that the hemA-encoded GTR reductase is a rate-limiting enzyme in the pathway (J.-M. Li, C. S. Russell, and S. D. Cosloy, Gene 82:2099-217, 1989). Increasing the copy number of hemM, whose product is believed to be required for efficient ALA formation (W. Chen, C. S. Russell, Y. Murooka, and S. D. Cosloy, J. Bacteriol. 176:2743-2746, 1994; M. Ikemi, K. Murakami, M. Hashimoto, and Y. Murooka, Gene 121:127-132, 1992), had no effect on either ALA pools or the rate of rHb1.1 accumulation. The hemA-encoded GTR reductase was found to be regulated by ALA. Some of our results differ from those reported by Hart and coworkers (R. A. Hart, P. T. Kallio, and J. E. Bailey, Appl. Environ. Microbiol. 60:2431-2437, 1994), who concluded that ALA formation is not the rate-limiting step in E. coli cells expressing Vitreoscilla hemoglobin.
Asunto(s)
Aldehído Oxidorreductasas/metabolismo , Ácido Aminolevulínico/metabolismo , Proteínas de la Membrana Bacteriana Externa , Proteínas Portadoras , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Hemo/biosíntesis , 5-Aminolevulinato Sintetasa/genética , 5-Aminolevulinato Sintetasa/metabolismo , Aldehído Oxidorreductasas/genética , Ácido Aminolevulínico/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Hemo/metabolismo , Hemo/farmacología , Chaperonas Moleculares , Regiones Promotoras GenéticasRESUMEN
Serum IgE directed against Der f 1, a protease found in the feces of Dermatophagoides farinae, correlates well with allergic sensitization to house dust mite in humans and is a risk factor for developing asthma. Native Der f 1 (nDer f 1) is produced as a pre-pro form and processed to an approximately 25-kDa mature form. We have expressed recombinant forms of Der f 1 (rDer f 1) in Pichia pastoris using AOX1-promoter expression vectors. Fusion of either the pro-enzyme form or the mature form to the Saccharomyces cerevisiae alpha factor pre-pro sequence resulted in secretion of the mature form of the protein from P. pastoris. The secreted protein was heterogeneously glycosylated at a single N-glycosylation site and had an apparent molecular mass of 35-50 kDa. Both the alpha factor signal peptide and the pro-enzyme region were efficiently processed during secretion. A version of the pro-enzyme with a mutated consensus N-linked glycosylation site was secreted from P. pastoris as a mature, unglycosylated, approximately 25-kDa protein. The IgE binding activity of this unglycosylated rDer f 1 was similar to that of glycosylated forms produced by P. pastoris and to nDer f 1 obtained from mites. Thus, oligosaccharides are not required for secretion from P. pastoris or for IgE binding in vitro. Recombinant and native versions of Der f 1 displayed protease activity on casein zymogram gels. The availability of a highly purified recombinant Der f 1 will facilitate experimental and clinical studies of mite allergy.